Methods and compositions for the diagnosis of sepsis using gamma peptide nucleic acids

ABSTRACT

The present disclosure provides for compositions of γPNA probes. Additionally, the present disclosure provide for methods and kits using γPNA probes for the diagnosis of sepsis.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/402,028 filed May 17, 2013, which is the U.S. National Stage filingof International Application No. PCT/US2013/041628, with aninternational filing date of May 17, 2013, which claims the benefit ofand priority to U.S. Provisional Application No. 61/649,342, filed May20, 2012 and U.S. Provisional Application No. 61/799,772, filed on Mar.15, 2013, the contents of which are incorporated by reference herein intheir entirety.

FIELD OF THE DISCLOSURE

This disclosure generally relates to methods, compositions, and kitsthat use peptide nucleic acid (PNA) as probes for the diagnosis ofsepsis.

BACKGROUND

Sepsis, a spectrum of severe immune disorders triggered by systemicinfections, is a leading cause of morbidity and mortality. In the U.S.alone, 751,000 sepsis cases occur annually, leading to 210,000mortalities and an economic burden of $24B. The primary causes of sepsisare usually symptomatic bacteremia or fungemia, a diagnosis that canonly be determined by laboratory testing. Detection of the infectingpathogen is essential to identifying patients and initiating the properantimicrobial therapy to avert or lessen a sepsis reaction.Traditionally, the first step in this process is a time-intensive stepof culturing the unknown pathogen from a patient specimen for a periodof 1-5 days. The culturing step results in a delay of effectivetreatment just at the earliest time of infection, which is a crucialtherapeutic window when therapy has the maximum benefit. Each singlehour delay in proper treatment increases the probability of mortality by7.6%.

SUMMARY

The present disclosure describes methods for diagnosing sepsis that donot require the time-consuming culturing step. In one embodiment, themethod comprises first contacting a plurality of γPNA capture probes togenomic material in a clinical sample obtained from a subject suspectedof having sepsis, wherein the γPNA capture probes comprise at least onesequence from one or more of the groups of probes selected from thegroup consisting of: SEQ ID NOS: 1-18 (provided in Table 1), SEQ ID NOS:19-22 (provided in Table 2), SEQ ID NOS: 23-28 (provided in Table 3),SEQ ID NOS: 29-34 (provided in Table 4), SEQ ID NOS: 35-38 (provided inTable 5), SEQ ID NOS: 39-57 (provided in Table 6), SEQ ID NOS: 58-72(provided in Table 7), SEQ ID NOS: 73-91 (provided in Table 8), SEQ IDNOS: 92-94 (provided in Table 9), SEQ ID NOS: 95-97 (provided in Table10), SEQ ID NOS: 98-110 (provided in Table 11), SEQ ID NOS: 111-113(provided in Table 12), SEQ ID NOS: 114-117 (provided in Table 13), SEQID NOS: 118-119 (provided in Table 14), SEQ ID NOS: 120-121 (provided inTable 15), SEQ ID NOS: 122-153 (provided in Table 16), SEQ ID NOS:154-166 (provided in Table 17), SEQ ID NOS: 167-190 (provided in Table18), SEQ ID NOS: 191-193 (provided in Table 19), SEQ ID NOS: 194-196(provided in Table 20), SEQ ID NOS: 197-211 (provided in Table 21), SEQID NOS: 212-215 (provided in Table 22), SEQ ID NOS: 216-230 (provided inTable 23), complementary sequence thereof, and functional equivalentsthereof; followed by the steps of heating the γPNA capture probes andthe sample; invading a plurality of targeted sepsis-related genomicmaterial by the γPNA capture probes; and detecting a presence of one ormore targeted genomic material. Detection of the presence of targetedgenomic material indicates the presence of a sepsis infection.

In some embodiments, the detection of targeted genomic materialcomprises of adding a plurality of γPNA reporter probes, which compriseof at least one sequence from one or both of the groups of probes: SEQID NOS: 231-248 (provided in Table 24) and SEQ ID NOS: 249-309 (providedin Table 25), complementary sequence thereof, and functional equivalentsthereof; heating the γPNA capture probe, γPNA reporter probes, and thesample; and invading of the γPNA reporter probes to the targeted genomicmaterial, wherein the γPNA reporter probes are used to detect thetargeted genomic materials.

In some embodiments, the contacting step is preceded by an amplificationstep comprising an enzymatic amplification of the of targetedsepsis-related genomic material.

In some embodiments, the genomic material in the clinical specimen issheared.

In some embodiments, the γPNA capture probes are bound to a supportsubstrate. In some embodiments, a first carbon-linker, comprising of atleast three carbons, binds the γPNA capture probes to the supportsubstrate. In some embodiments, the support substrate is selected fromthe group consisting of: a magnetic bead, a bead, a well, a plate, forexample polystyrene microtiter plate, a test tube, a stick, for examplea dipstick, plastic, glass, and a chip or biochip. In some embodiments,the support substrate is coated with Avidin, Neutravidin, orStreptavidin.

In some embodiments, the γPNA capture probes and/or γPNA reporter probescomprise one or more functional moiety selected from the groupconsisting of a binding molecule, a spacer group, a linker group, ahydrophobicity-changing group, a charge-inducing group, and a structuralchange-inducing group. In some embodiments, the spacer group is selectedfrom the group consisting of: (ethylene)glycol, di(ethylene)glycol,tri(ethylene)glycol, poly(ethylene)glycol, 6-carbon linker, and 12carbon linker. In some embodiments, the linker group is selected fromthe group consisting of: COOH group, NETS-ester group, malemidechemistry, Click chemistry, streptavidin, and biotinylation. In someembodiments, the hydrophobicity-changing group is selected from thegroup consisting of: a naturally polar or charged side group or linkerthat decreases hydrophobicity, and a naturally apolar and uncharged sidegroup or linker that increases hydrophobicity. In some embodiments, thecharge-inducing group is selected from the group consisting of: COOHgroup, NH₃ groups, OH groups, and metallic ions. In some embodiments,the structural change-inducing group induces a chemical modificationalong the peptide backbone of PNA and is selected from the groupconsisting of: amino acid-based side chain, nanoparticle, small moleculeor intercalating agent. In some embodiments, the γPNA probe comprisesbiotin or hapten.

In some embodiments, detecting the presence of one or more targetedgenomic material is through a signal selected from the group consistingof: fluorescence, luminescence, FRET, colorimetric, calorimetric,interference patterns, pH, resistance/conductivity, enzymatic functionand kinetics, protein structure, and electrical potential.

In some embodiments, the γPNA reporter probes comprise a secondcarbon-linker. In some embodiments, the second carbon-linker comprisesof one or more biotinylation sites.

An alternative embodiment provides for a composition for diagnosingsepsis, wherein the composition comprises a γPNA probe compositioncomprising at least one sequence from one or more of the groups ofprobes selected from the group consisting of: SEQ ID NOS: 1-18, SEQ IDNOS: 19-22, SEQ ID NOS: 23-28, SEQ ID NOS: 29-34, SEQ ID NOS: 35-38, SEQID NOS: 39-57, SEQ ID NOS: 58-72, SEQ ID NOS: 73-91, SEQ ID NOS: 92-94,SEQ ID NOS: 95-97, SEQ ID NOS: 98-110, SEQ ID NOS: 111-113, SEQ ID NOS:114-117, SEQ ID NOS: 118-119, SEQ ID NOS: 120-121, SEQ ID NOS: 122-153,SEQ ID NOS: 154-166, SEQ ID NOS: 167-190, SEQ ID NOS: 191-193, SEQ IDNOS: 194-196, SEQ ID NOS: 197-211, SEQ ID NOS: 212-215, SEQ ID NOS:216-230, SEQ ID NOS: 231-248, SEQ ID NOS: 249-309, complementarysequence thereof, and functional equivalents thereof.

In some embodiments, the γPNA probe comprises a support substrate. Insome embodiments, the support substrate is selected from the groupconsisting of: a magnetic bead, a bead, a well, a plate, for examplepolystyrene microtiter plate, a test tube, a stick, for example adipstick, plastic, glass, and a chip or biochip. In some embodiments,the support substrate is coated with Avidin, Neutravidin, orStreptavidin.

In some embodiments, the γPNA probe comprises one or more functionalmoiety selected from the group consisting of a binding molecule, aspacer group, a linker group, a hydrophobicity-changing group, acharge-inducing group, and a structural change-inducing group.

In some embodiments, the γPNA probe emits a detectable signal selectedfrom the group consisting of: fluorescence, luminescence, FRET,colorimetric, calorimetric, interference patterns, pH,resistance/conductivity, enzymatic function and kinetics, proteinstructure, and electrical potential.

In some embodiments, the γPNA probe comprises a carbon-linker comprisingat least three carbons. In some embodiments, the carbon-linker comprisesof one or more biotinylation sites.

An alternative embodiment provides for a kit for detecting sepsiscomprising a γPNA capture probe composition comprising at least onesequence from one or more of the groups of probes selected from thegroup consisting of: SEQ ID NOS: 1-18, SEQ ID NOS: 19-22, SEQ ID NOS:23-28, SEQ ID NOS: 29-34, SEQ ID NOS: 35-38, SEQ ID NOS: 39-57, SEQ IDNOS: 58-72, SEQ ID NOS: 73-91, SEQ ID NOS: 92-94, SEQ ID NOS: 95-97, SEQID NOS: 98-110, SEQ ID NOS: 111-113, SEQ ID NOS: 114-117, SEQ ID NOS:118-119, SEQ ID NOS: 120-121, SEQ ID NOS: 122-153, SEQ ID NOS: 154-166,SEQ ID NOS: 167-190, SEQ ID NOS: 191-193, SEQ ID NOS: 194-196, SEQ IDNOS: 197-211, SEQ ID NOS: 212-215, SEQ ID NOS: 216-230, complementarysequence thereof, and functional equivalents thereof.

In some embodiments, the kit comprises a γPNA reporter probe compositioncomprising at least one sequence from one or both of the groups ofreporter probes: SEQ ID NOS: 231-248 and SEQ ID NOS: 249-309,complementary sequence thereof, and functional equivalents thereof.

In some embodiments, the γPNA capture probes are bound to a supportsubstrate. In some embodiments, the support substrate is selected fromthe group consisting of: a magnetic bead, a bead, a well, a plate, forexample polystyrene microtiter plate, a test tube, a stick, for examplea dipstick, plastic, glass, and a chip or biochip. In some embodiments,the support substrate is coated with Avidin, Neutravidin, orStreptavidin.

In some embodiments, the γPNA probe composition emits a detectablesignal selected from the group consisting of: fluorescence,luminescence, FRET, colorimetric, calorimetric, interference patterns,pH, resistance/conductivity, enzymatic function and kinetics, proteinstructure, and electrical potential.

Another alternative embodiment provides for a method for diagnosingsepsis comprising contacting a plurality of γPNA reporter probes togenomic material in a clinical sample obtained from a subject suspectedof having sepsis, wherein the γPNA reporter probes comprise at least onesequence from one or both of the groups of reporter probes: SEQ ID NOS:231-248 and SEQ ID NOS: 249-309, complementary sequence thereof, andfunctional equivalents thereof; heating the γPNA reporter probes and thesample; invading a plurality of targeted sepsis-related genomic materialby the γPNA reporter probes; contacting the plurality of sepsis-relatedgenomic material with γPNA capture probes, wherein the γPNA captureprobes comprise at least one sequence from one or more of the groups ofprobes selected from the group consisting of: SEQ ID NOS: 1-18, SEQ IDNOS: 19-22, SEQ ID NOS: 23-28, SEQ ID NOS: 29-34, SEQ ID NOS: 35-38, SEQID NOS: 39-57, SEQ ID NOS: 58-72, SEQ ID NOS: 73-91, SEQ ID NOS: 92-94,SEQ ID NOS: 95-97, SEQ ID NOS: 98-110, SEQ ID NOS: 111-113, SEQ ID NOS:114-117, SEQ ID NOS: 118-119, SEQ ID NOS: 120-121, SEQ ID NOS: 122-153,SEQ ID NOS: 154-166, SEQ ID NOS: 167-190, SEQ ID NOS: 191-193, SEQ IDNOS: 194-196, SEQ ID NOS: 197-211, SEQ ID NOS: 212-215, SEQ ID NOS:216-230, complementary sequence thereof, and functional equivalentsthereof; heating the γPNA reporter probes, the γPNA capture probes, andthe sample; invading the plurality of targeted sepsis-related genomicmaterial by the γPNA capture probes; and detecting a presence of one ormore targeted genomic material, wherein detection of the presence oftarget genomic material is indicative of sepsis infection.

In some embodiments, the support substrate is selected from the groupconsisting of: a magnetic bead, a bead, a well, a plate, for examplepolystyrene microtiter plate, a test tube, a stick, for example adipstick, plastic, glass, and a chip or biochip. In some embodiments,the support substrate is coated with Avidin, Neutravidin, orStreptavidin.

In some embodiments, wherein the γPNA capture probes and γPNA reporterprobes comprise one or more functional moiety selected from the groupconsisting of: a binding molecule, a spacer group, a linker group, ahydrophobicity-changing group, a charge-inducing group, and a structuralchange-inducing group.

In some embodiments, the γPNA capture probes and γPNA reporter probescomprise biotin or hapten.

In some embodiments, the γPNA reporter probes emit a detectable signalselected from the group consisting of: fluorescence, luminescence, FRET,colorimetric, calorimetric, interference patterns, pH,resistance/conductivity, enzymatic function and kinetics, proteinstructure, and electrical potential.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic of γPNA capture probes binding to a magnetic beadthat is coated with neutravidin.

FIG. 1B is a schematic of target genomic materials binding to γPNAcapture probes.

FIG. 1C is a schematic of γPNA reporter probes binding to immobilizedtarget genomic materials.

FIG. 1D is a schematic of reporter enzyme conjugates binding to γPNAreporter probes.

FIG. 1E is a schematic of adding enzyme substrate to induce a signal.

FIG. 2 is a gel shift assay demonstrating the specificity of γPNAprobes.

FIG. 3 is a gel shift assay demonstrating the specificity of γPNAcapture probes for different species of the same genus.

DETAILED DESCRIPTION

It is to be appreciated that certain aspects, modes, embodiments,variations and features of the invention are described below in variouslevels of detail in order to provide a substantial understanding of thepresent invention. The definitions of certain terms as used in thisspecification are provided below. Unless defined otherwise, alltechnical and scientific terms used herein generally have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. As used herein the following terms havethe following meanings.

Definitions

As used herein, the term “comprising” or “comprises” is intended to meanthat the compositions and methods include the recited elements, but notexcluding others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the stated purpose. Thus,a composition consisting essentially of the elements as defined hereinwould not exclude other materials or steps that do not materially affectthe basic and novel characteristic(s) of the claimed invention.“Consisting of” shall mean excluding more than trace elements of otheringredients and substantial method steps. Embodiments defined by each ofthese transition terms are within the scope as described herein.

The term “about” when used before a numerical designation, e.g.,temperature, time, amount, and concentration, including range, indicatesapproximations which may vary by (+) or (−) 10%, 5%, or 1%.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a cell” includesa combination of two or more cells, and the like.

As used herein, “sample” refers to blood samples, culture samples, orDNA samples that originate from or are generated from a person orpatient that is suspected of having sepsis. The samples taken or derivedfrom the person or patient are believed to contain pathogenic genomicmaterial related to sepsis. Additionally, sample can refer to mixture ofgenomic material that is to be tested for the presence sepsis relatedpathogenic materials.

As used herein, “pathogenic or bacterial genomic material” or “targetedsepsis-related genomic material” or “targeted genomic material” refer toDNA, RNA, oligonucleotides, or polynucleic acids related to any genus orspecies of pathogens related to sepsis. Additionally, DNA and RNA arebroadly used to include, but not limited to, ribosomal DNA and RNA (rDNAand rRNA), messenger RNA (mRNA), transfer DNA (tDNA), and mitochondrialDNA (mDNA). Examples of such pathogens include, but are not limited to,Staphylococcus epidermidis, Staphylococcus aureus, Enterococcusfaecalis, Enterococcus faecium, and Escherichia coli.

As used herein, “support substrate” refers a solid substrate upon whichγPNAs can be immobilized. In one embodiment, the support substrate is amagnetic bead, a bead, a well, a plate, for example polystyrenemicrotiter plate, a test tube, a stick, for example a dipstick, plastic,glass, and a chip or biochip. In some embodiments, the support substrateis silicon based or coated with either a semiconductive, conductive, orinsulating material. In some embodiments, the support substrate includesmetallic surfaces that are functionalized. In other embodiments thesolid substrate may be manufactured from polymers, nylon,nitrocellulose, polyacrylamide, oxides. In some embodiments, the solidsupport is manufactured from multiple materials. In some embodiments,the surface of the support substrate is coated with an aminosilane orany other commonly known surface treatments, such as epoxysilanes.

As used herein, “immobilizing” describes binding of γPNA capture probesonto a solid, support substrate prior to the introduction of a sample.Immobilization of the γPNA capture probes sequences on a supportsubstrate can be achieved through various means such as covalent bindingprotocols or non-covalent binding protocols. Binding modalities andchemistries are commonly known to those skilled in the art.

As used herein, “signal” refers to that which is detectable thoughoptical modalities, or through electrical modalities, or throughbiological modalities, or through chemical modalities.

As used herein, “diagnosis” refers to determining or identifying thepresence or absence of one or more sepsis-inducing pathogen in apatient. Additionally, diagnosis can also refer to determining apatient's susceptibility to sepsis.

As used herein, “washing” refers to steps to remove unwanted, unbound,weakly bound, non-specifically bound genomic and other non-genomicmaterial from the vicinity of the PNA probe. Washing steps are wellestablished within the field and have been optimized for numerousbiological assays such as ELISA, Western blot assays, Southern blotassays, Northern Blot assays, DNA microarrays, RNA microarrays, proteinmicroarrays, and LiPA. Washing steps, and optimizing of the washingsteps are well established and known to those skilled in the art.

The term “invasion” or “invade” refers to γPNA probes, both capture andreporter, binding to target sequences using either natural or inducedstructural fluctuations, referred to as DNA breathing or DNA bubbles.Nucleic acids may, on occasion, present their nucleobases to the bulk,meaning the nucleobases are not hidden within the structure. When thisoccurs, the γPNA, may bind to those exposed nucleobases through typicalWatson Crick base-pairing rules. Upon the closure of this DNA bubble,the γPNA remains bound to the nucleic acid region effectivelydisplacing, locally, the complementary nucleic acid strand—hence γPNAinvades the nucleic acid structure locally.

Culture-free diagnostic tools are required for the timely and propertreatment of microbial pathogens which induce a septic response inafflicted patients. While recent advances in molecular diagnostics haverevolutionized numerous disease areas in clinical testing, severetechnical limitations prevent molecular techniques from havingsignificant impact in cases of bacteremia or fungemia. Identification ofthe infecting pathogen and its susceptibility to antimicrobial therapystill require a time-intensive step of culturing. Thus, the realities ofsepsis remain grim; a mortality rate of 28% with 210,000 annual deathsin the U.S. alone. The present disclosure describes compounds, methods,and kits related to a culture-free approach to sepsis pathogenidentification and susceptibility analysis.

Peptide Nucleic Acid

Peptide nucleic acids (PNAs) are synthetic, or non-naturally, occurringoligomers, which have displayed the ability to bind to both DNA and RNAaccording to either Watson-Crick and/or Hoogstgein base pairing. In PNA,the negatively charged sugar-phosphate backbone of natural DNA or RNAhas been replaced with a neutral peptide backbone. PNA's neutralbackbone negates the energy penalty natural probes must expend toovercome the mutual repulsion of their negatively charged phosphatebackbone. Thus, PNA binds to nucleic acids (NAs) with much greateraffinities than natural probes. Other advantages of PNAs in generalinclude: the ability to bind to both natural and synthetic targets, fastbinding kinetics, and the ability to add chemical moieties such as, butnot limited to, fluorescent dyes, biotin, protein binding agents,radio-labeling, or quantum dots.

A new class of PNA, termed γPNA, is PNA with a simple backbonemodification at the γ-position of the N-(2-aminoethyl) glycine backbonethat generates a chiral center. In an unbound state, the configurationof ordinary PNA or DNA probes is a random, globular structure. Incontrast, unbound γPNA probes assume a right-handed helix structure,pre-organized for Watson-Crick base pairing, which greatly facilitatingbinding.

γPNA probes have several major advantages of over natural DNA probes andordinary PNA probes. Some of the advantages include:

-   -   1) γPNA has substantially greater affinity to nucleic acids than        other natural or synthetic probes. Typical Tm values for        γPNA/DNA 15 bp hybrids are ˜20° C. higher (Tm>95° C.) than        equivalent ordinary PNA/DNA hybrids and ˜50° C. higher than        natural dsDNA. γPNA Kd values are even comparable to antibodies        (nM-pM).    -   2) γPNA is significantly more sequence specific. Ordinary PNAs        are commonly used as primer clamps to overcome the poor sequence        specificity of natural DNA by removing the likelihood of PCR        beacons binding to slightly mismatched regions. γPNAs have even        greater sequence specificity than ordinary PNAs, as reflected in        the greater increase in ΔTm when a mismatch is induced.    -   3) γPNA has the unique ability to invade structured nucleic        acids, such as double stranded DNA (dsDNA) and RNA, which        facilitates a less complex method for target identification.        γPNA's affinity to single-stranded NAs is so high that it has        the ability to naturally invade the double stranded structure        and bind through standard Watson-Crick base pairing to the        correct sequence. Binding to single-stranded NAs occurs just as        it would for other synthetic and natural nucleic acid probes.        PNA Targets and Sequences

Comparative analysis of ribosomal DNA (rDNA) sequences has become awell-established method for establishing phylogenetic relationshipsbetween microbial species. Microbial rDNA are among the most highlyconserved and most rigorously studied regions in a microbial genome.Minute differences in rDNA sequences enable the design of highlyspecific probes for target regions, which are specific to one or morepathogens.

Polymicrobial infections are problematic in that numerous pathogens withtheir own inherent resistance traits induce similar pathophysiologicaltraits in the host during sepsis. Despite the similarity in hostresponse and the high similarity at the genomic level of numerouspathogens; treatment regimens could vary significantly depending onspecies traits as well as the presence or absence of genes which encodefor antimicrobial resistance.

Sequence analysis for specific genes that might encode for a resistanceto a particular antimicrobial compound has likewise been established.Multiple regions in these genes are highly conserved and can be used tocreate markers for targeting. The targeting the highly conserved regionswould enable the detection of a gene sequence that encodes forantimicrobial resistance.

The γPNA probes preferably target rDNA/rRNA sequence of the microbialpathogen. In the present disclosure, the γPNA probe sequences all relateto identifying bacterium involved in sepsis. Tables 1-25 describes anon-exclusive list of sequences capable of identifying bacteriuminvolved with sepsis. Since the γPNA probes will bind to dsDNA, oneskilled in the art would know that the reverse-complementary sequencesof the sequences in Tables 1-25 can also be γPNA probe sequences. Thus,in some embodiments, the target sequence may be thereverse-complementary sequence to those identified here.

In some embodiments, γPNA probe sequences are those which will bind tothe corresponding rDNA/rRNA target sequences through Watson-Crickbase-pairing. Additional base-pairing methods such as Hoogstein havebeen demonstrated with other PNA variants (such as bis-PNA).

Since PNAs do not have phosphate-sugar backbone, orientation is guidedby the terminus of the peptide backbone for proper binding. Therefore,the C-terminus aligns with the 5′ end of the DNA/RNA target, and theN-terminus aligns with the 3′ end of the DNA/RNA target.

In addition to the natural nucleobases, the inclusion of modified orsynthetic nucleobases may also be included to enhance γPNAcharacteristics. A common synthetic nucleobase for use with γPNA istypically called the ‘G-clamp’ which refers to a pseudo-cytosine(9-(2-guanidinoethoxy) phenoxazine). Another common synthetic nucleobaseused in PNAs is the J-base which carries a hydrogen atom at the N3position allowing its Hoogsteen pairing with a guanine base withoutprotonation.

TABLE 1 γPNA capture probe sequences for Staphylococcus aureus SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 1 CGG AAC ATC TTC TTC SEQID NO: 2 TCA GAA GAT GCG GAA SEQ ID NO: 3 CCT GAT AAG CGT GAG SEQ ID NO:4 GAG TCC ACT TAG GCC SEQ ID NO: 5 CAC TTA GGC CCA CCA SEQ ID NO: 6 AGGCCC ACC ATT ATT SEQ ID NO: 7 AAC GGA CGA GAA GCT SEQ ID NO: 8 TCC TTTGAC AAC TCT SEQ ID NO: 9 AAC GGA CGA GAA GCT SEQ ID NO: 10 AGA GAT AGAGCC TTC SEQ ID NO: 11 TTT GAC AAC TCT AGA SEQ ID NO: 12 CTT CTC TGA TGTTAG SEQ ID NO: 13 GGA TAA TAT TTT GAA SEQ ID NO: 14 TTC AAA AGT GAA AGASEQ ID NO: 15 AGA CGG TCT TGC TGT SEQ ID NO: 16 ATC CGC GCT GCA TTA SEQID NO: 17 AGA ACA TAT GTG TAA SEQ ID NO: 18 TAA CCT TTT AGG AGC

TABLE 2 γPNA capture probe sequences for Enterococcus faecalis SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 19 ACA AGG ACG TTA GTASEQ ID NO: 20 CTT TCC TCC CGA GTG SEQ ID NO: 21 CCT ACC CAT CAG AGG SEQID NO: 22 GGA CGT TAG TAA CTG

TABLE 3 γPNA capture probe sequences for Enterococcus faecium SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 23 CTT GCT CCA CCG GAASEQ ID NO: 24 CTT TTT CCA CCG GAG SEQ ID NO: 25 ATG GTT TTG ATT TGA SEQID NO: 26 CTT TTT CCA CCG GAG SEQ ID NO: 27 CGT ATA ACA ATC GAA SEQ IDNO: 28 CGT ATA ACA ATC AAA

TABLE 4 γPNA capture probe sequences for Escherichia coli SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 29 AAC AGG AAG AAG CTTSEQ ID NO: 30 GGA GTA AAG TTA ATA SEQ ID NO: 31 ATA CCT TTG CTC ATT SEQID NO: 32 CAT CTG ATA CTG GCA SEQ ID NO: 33 TTG CTT CTT TGC TGA SEQ IDNO: 34 AGC TTG AGT CTC GTA

TABLE 5 γPNA capture probe sequences for Staphylococcus epidermidis SEQID Sequence (N-terminus to C-terminus) SEQ ID NO: 35 GCT CCT CTG ACG TTASEQ ID NO: 36 ATA ATA TAT TGA ACC SEQ ID NO: 37 TTC AAT AGT GAA AGA SEQID NO: 38 AAC TAT GCA CGT CTT

TABLE 6 γPNA capture probe sequences for Pseudomonas aeruginosa SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 39 GAG CGG ATG AAG GGASEQ ID NO: 40 CTT GCT CCT GGA TTC SEQ ID NO: 41 AAT CTG CCT GGT AGT SEQID NO: 42 ATA ACG TCC GGA AAC SEQ ID NO: 43 CCG CAT ACG TCC TGA SEQ IDNO: 44 AGA TGA GCC TAG GTC SEQ ID NO: 45 GAC GAT CCG TAA CTG SEQ ID NO:46 CAG TAA GTT AAT ACC SEQ ID NO: 47 CAA CAG AAT AAG CAC SEQ ID NO: 48TCC AAA ACT ACT GAG SEQ ID NO: 49 CTG AGC TAG AGT ACG SEQ ID NO: 50 AATTTC CTG TGT AGC SEQ ID NO: 51 GCG TAG ATA TAG GAA SEQ ID NO: 52 ACC ACCTGG ACT GAT SEQ ID NO: 53 TGT CGA CTA GCC GTT SEQ ID NO: 54 AGC CGT TGGGAT CCT SEQ ID NO: 55 TGA GAT CTT AGT GGC SEQ ID NO: 56 AAC TCA GAC ACAGGT SEQ ID NO: 57 TTG TCC TTA GTT ACC

TABLE 7 γPNA capture probe sequences for Streptococcus pneumoniae SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 58 CTG AAG GAG GAG CTTSEQ ID NO: 59 AGG AGC TTG CTT CTC SEQ ID NO: 60 ATG ACA TTT GCT TAA SEQID NO: 61 ACT TGC ATC ACT ACC SEQ ID NO: 62 AAT GGA CGG AAG TCT SEQ IDNO: 63 AAG AAC GAG TGT GAG SEQ ID NO: 64 AAA GTT CAC ACT GTG SEQ ID NO:65 TAT CTT ACC AGA AAG SEQ ID NO: 66 TTA GAT AAG TCT GAA SEQ ID NO: 67AAA GGC TGT GGC TTA SEQ ID NO: 68 TTA ACC ATA GTA GGC SEQ ID NO: 69 AAACTG TTT AAC TTG SEQ ID NO: 70 ACT TGA GTG CAA GAG SEQ ID NO: 71 TCT CTGGCT TGT AAC SEQ ID NO: 72 CCT CTG ACC GCT CTA

TABLE 8 γPNA capture probe sequences for Streptococcus pyogenes SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 73 AGA ACT GGT GCT TGCSEQ ID NO: 74 CTG GTG CTT GCA CCG SEQ ID NO: 75 TTG CAC CGG TTC AAG SEQID NO: 76 TAA CCT ACC TCA TAG SEQ ID NO: 77 ATA AGA GAG ACT AAC SEQ IDNO: 78 AGA CTA ACG CAT GTT SEQ ID NO: 79 AGT AAT TTA AAA GGG SEQ ID NO:80 AAT TGC TCC ACT ATG SEQ ID NO: 81 CTC CAC TAT GAG ATG SEQ ID NO: 82TTA GAG AAG AAT GAT SEQ ID NO: 83 GAA AAT CCA CCA AGT SEQ ID NO: 84 TGACGG TAA CTA ACC SEQ ID NO: 85 AAA GGC ATT GGC TCA SEQ ID NO: 86 CTC AACCAA TGT ACG SEQ ID NO: 87 AAA CTG GAG AAC TTG SEQ ID NO: 88 CTT GAG TGCAGA AGG SEQ ID NO: 89 GCT TAG TGC CGG AGC SEQ ID NO: 90 ATA GAG TTT TACTTC SEQ ID NO: 91 GGT ACA TCG GTG ACA

TABLE 9 γPNA capture probe sequences for Klebsiella pneumonia SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 92 AAG GCG ATA AGG TTASEQ ID NO: 93 TGC CAG CGG TTA GGC SEQ ID NO: 94 AAG GCG ATG AGG TTA

TABLE 10 γPNA capture probe sequences for Enterobacter species SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 95 AAG GCG TTA AGG TTASEQ ID NO: 96 ATA ACC TTG GCG ATT SEQ ID NO: 97 TGC CAG CGG TCC GGC

TABLE 11 γPNA capture probe sequences for Proteus mirabilis SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 98 AAC AGG AGA AAG CTTSEQ ID NO: 99 TTT CTT GCT GAC GAG SEQ ID NO: 100 GGA TCT GCC CGA TAG SEQID NO: 101 ATA ATG TCT ACG GAC SEQ ID NO: 102 TAC GGA CCA AAG CAG SEQ IDNO: 103 TTG CAC TAT CGG ATG SEQ ID NO: 104 CGG ATG AAC CCA TAT SEQ IDNO: 105 AAT ACC CTT GTC AAT SEQ ID NO: 106 TCA ATT AAG TCA GAT SEQ IDNO: 107 ATC TGA AAC TGG TTG SEQ ID NO: 108 ATT TAG AGG TTG TGG SEQ IDNO: 109 TTG TGG TCT TGA ACC SEQ ID NO: 110 AGC GAA TCC TTT AGA

TABLE 12 γPNA capture probe sequences for Staphylococcus lugdunensis SEQID Sequence (N-terminus to C-terminus) SEQ ID NO: 111 GAC TGG GAC AACTTC SEQ ID NO: 112 ATA ATA TGT TGA ACC SEQ ID NO: 113 GTC TTA GGA TCGTAA

TABLE 13 γPNA capture probe sequences for Staphylococcus warneri SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 114 GGA TAA CAT ATT GAASEQ ID NO: 115 AAA GGC GGC TTT GCT SEQ ID NO: 116 TCT GTT ATC AGG GAASEQ ID NO: 117 GTA CCT GAT CAG AAA

TABLE 14 γPNA capture probe sequences for Staphylococcus hominis SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 118 AGA TGG CTT TGC TATSEQ ID NO: 119 GAG ATA GAA GTT TCC

TABLE 15 γPNA capture probe sequences for Serratia Marcescens SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 120 AAG GTG GTG AGC TTASEQ ID NO: 121 TTA ATA CGT TCA TCA

TABLE 16 γPNA capture probe sequences for Acinetobacter baumannii SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 122 AAG GTA GCT TGC TACSEQ ID NO: 123 TTG CTA CCG GAC CTA SEQ ID NO: 124 AAT GCT TAG GAA TCTSEQ ID NO: 125 AAT CTG CCT ATT AGT SEQ ID NO: 126 ACA ACA TCT CGA AAGSEQ ID NO: 127 AAA GGG ATG CTA ATA SEQ ID NO: 128 ACC TTG CGC TAA TAGSEQ ID NO: 129 ATG AGC CTA AGT CGG SEQ ID NO: 130 CGA TCT GTA GCG GGTSEQ ID NO: 131 AAC CCT GAT CCA GCC SEQ ID NO: 132 AGG CTA CTT TAG TTASEQ ID NO: 133 TTT AGT TAA TAC CTA SEQ ID NO: 134 TAC CTA GAG ATA GTGSEQ ID NO: 135 ATA GTG GAC GTT ACT SEQ ID NO: 136 CAG CCA TCT GGC CTASEQ ID NO: 137 GCC TAA TAC TGA CGC SEQ ID NO: 138 TCT ACT AGC CGT TGGSEQ ID NO: 139 CCT TTG AGG CTT TAG SEQ ID NO: 140 CGA TAA GTA GAC CGCSEQ ID NO: 141 GTC GCA AGA CTA AAA SEQ ID NO: 142 TGG CCT TGA CAT ACTSEQ ID NO: 143 ATA CTA GAA ACT TTC SEQ ID NO: 144 AAT CTA GAT ACA GGTSEQ ID NO: 145 TTT TCC TTA CTT GCC SEQ ID NO: 146 CCA GCA TTT CGG ATGSEQ ID NO: 147 ACT TTA AGG ATA CTG SEQ ID NO: 148 TTG CTA CAC AGC GATSEQ ID NO: 149 ATG TGA TGC TAA TCT SEQ ID NO: 150 TAA TCT CAA AAA GCCSEQ ID NO: 151 AAG CCG ATC GTA GTC SEQ ID NO: 152 AAT GCC GCG GTG AATSEQ ID NO: 153 TAG CCT AAC TGC AAA

TABLE 17 γPNA capture probe sequences for Stenotrophomonas maltophiliaSEQ ID Sequence (N-terminus to C-terminus) SEQ ID NO: 154 AAC GGC AGCACA GTA SEQ ID NO: 155 TAA GAG CTT GCT CTT SEQ ID NO: 156 GAA TAC ATCGGA ATC SEQ ID NO: 157 AAA CTT ACG CTA ATA SEQ ID NO: 158 ATC CAG CTGGTT AAT SEQ ID NO: 159 GTA CCC AAA GAA TAA SEQ ID NO: 160 TTG TTT AAGTCT GTT SEQ ID NO: 161 AGC TAC CTG GAC CAA SEQ ID NO: 162 TGC AAT TTGGCA CGC SEQ ID NO: 163 AAC GCG TTA AGT TCG SEQ ID NO: 164 CTG CAA GCCGGC GAC SEQ ID NO: 165 AGA AAC CCT ATC TCA SEQ ID NO: 166 AGC ATT GCTGCG GTG

TABLE 18 γPNA capture probe sequences for Capture Coagulase-negativestaphylococci (CoNS). Sequence Pool # SEQ ID (N-terminus to C-terminus)1 SEQ ID NO: 167 AAC AGA TAA GGA GCT SEQ ID NO: 168 AAC AGA CGA GGA GCTSEQ ID NO: 169 AAC AGA CAA GGA GCT 2 SEQ ID NO: 170 CTC CTT TGA CGT TAGSEQ ID NO: 171 CTC CTC TGA CGT TAG SEQ ID NO: 172 CTC TTT TGA CGT TAGSEQ ID NO: 173 CTT CTC TGA CGT TAG 3 SEQ ID NO: 174 GGA TAA TAT TTC GAASEQ ID NO: 175 GGA TAA CAT ATT GAA SEQ ID NO: 176 GGA TAA TAT ATT GAASEQ ID NO: 177 GGA TAA TAT GTT GAA 4 SEQ ID NO: 178 AGA TGG TTT TGC TATSEQ ID NO: 179 AGG CGG CTT TGC TGT SEQ ID NO: 180 AGA CGG TTT TGC TGTSEQ ID NO: 181 AGA TGG CTT TGC TAT 5 SEQ ID NO: 182 ACC CGC GCC GTA TTASEQ ID NO: 183 ACC TGC GCC GTA TTA SEQ ID NO: 184 ATC CGC GCC GTA TTASEQ ID NO: 185 ATC CGC GCC GCA TTA 6 SEQ ID NO: 186 AGA ACA TAC GTG TAASEQ ID NO: 187 AGA ACA AAC GTG TAA SEQ ID NO: 188 AGA ACA AAT GTG TAA 7SEQ ID NO: 189 TAA CCA TTT GGA GCT SEQ ID NO: 190 TAA CCA TTT ATG GAG

TABLE 19 γPNA capture probe sequences for Candida tropicalis SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 191 AAT GTC TTC GGA CTCSEQ ID NO: 192 CAT CTT TCT GAT GCG SEQ ID NO: 193 GGC TAG CCT TTT GGC

TABLE 20 γPNA capture probe sequences for Candida parapsilosis SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 194 ATC TTT TTT GAT GCGSEQ ID NO: 195 TGG CTA GCC TTT TTG SEQ ID NO: 196 TAT TCA GTA GTC AGA

TABLE 21 γPNA capture probe sequences for Candida glabrata SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 197 CTT TAC TAC ATG GTASEQ ID NO: 198 ATG GTA TAA CTG TGG SEQ ID NO: 199 ATG CTT AAA ATC TCGSEQ ID NO: 200 TCC GAT TTT TTC GTG SEQ ID NO: 201 TGT ACT GGA ATG CACSEQ ID NO: 202 AAC CCC AAG TCC TTG SEQ ID NO: 203 TTG TGG CTT GGC GGCSEQ ID NO: 204 ACG TTT GGT TCT ATT SEQ ID NO: 205 TAT TCA ATT GTC AGASEQ ID NO: 206 TGT TTT TTT AGT GAC SEQ ID NO: 207 TAA ATA GTG GTG CTASEQ ID NO: 208 ATT TGC TGG TTG TCC SEQ ID NO: 209 TAT CGG TTT CAA GCCSEQ ID NO: 210 AGC GAG TCT AAC CTT SEQ ID NO: 211 TCT TGG TAA TCT TGT

TABLE 22 γPNA capture probe sequences for Candida albicans SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 212 TTC TGG GTA GCC ATTSEQ ID NO: 213 GCC ATT TAT GGC GAA SEQ ID NO: 214 GGT AGC CAT TTA TGGSEQ ID NO: 215 CTA TCG ACT TCA AGT

TABLE 23 γPNA capture probe sequences for Aspergillus species SEQ IDSequence (N-terminus to C-terminus) SEQ ID NO: 216 TAC CTT ACT ACA TGGSEQ ID NO: 217 ACT ACA TGG ATA CCT SEQ ID NO: 218 TGC TAA AAA CCT CGASEQ ID NO: 219 TAA AAA ACC AAT GCC SEQ ID NO: 220 TAA CGA ATC GCA TGGSEQ ID NO: 221 TAC CAT GGT GGC AAC SEQ ID NO: 222 AAT CTA AAT CCC TTASEQ ID NO: 223 AGT ACT GGT CCG GCT SEQ ID NO: 224 GAA CCT CAT GGC CTTSEQ ID NO: 225 GCC TTC ACT GGC TGT SEQ ID NO: 226 TTT CTA TGA TGA CCCSEQ ID NO: 227 TCG GCC CTT AAA TAG SEQ ID NO: 228 GAG TAC ATC ACC TTGSEQ ID NO: 229 CTT GTT AAA CCC TGT SEQ ID NO: 230 AGC TCG TGC CGA TTA

TABLE 24 γPNA reporter probe sequences for bacteria SEQ ID Sequence(N-terminus to C-terminus) SEQ ID NO: 231 GGA CTA CCA GGG TAT SEQ ID NO:232 ACC AGG GTA TCT AAT SEQ ID NO: 233 CGG GAA CGT ATT CAC SEQ ID NO:234 CAG CAG CCG CGG TAA SEQ ID NO: 235 ATG TGG TTT AAT TCG SEQ ID NO:236 GT GCC AGC AGC CGC SEQ ID NO: 237 AAC GAG CGC AAC CC SEQ ID NO: 238GTG GTT TAA TTC GA SEQ ID NO: 239 ACC TTG TTA CGA CTT SEQ ID NO: 240 CGACAG AGT TTG ATC SEQ ID NO: 241 ACC TTG TTA CGA CTT SEQ ID NO: 242 CAGCCG CGG TAA TAC SEQ ID NO: 243 AAC AGG ATT AGA TAC SEQ ID NO: 244 GTCGTC AGC TCG TGT SEQ ID NO: 245 ATG TTG GGT TAA GTC SEQ ID NO: 246 GAATCG CTA GTA ATC SEQ ID NO: 247 CTT GTA CAC ACC GCC SEQ ID NO: 248 GGACTA CCA GGG TAT CTA AT

TABLE 25 γPNA reporter probe sequences for fungi SEQ ID Sequence(N-terminus to C-terminus) SEQ ID NO: 249 GTG AAA CTG CGA ATG SEQ ID NO:250 CTG CGA ATG GCT CAT SEQ ID NO: 251 GCT CAT TAA ATC AGT SEQ ID NO:252 TCA GTT ATC GTT TAT SEQ ID NO: 253 CGT TTA TTT GAT AGT SEQ ID NO:254 TCT AGA GCT AAT ACA SEQ ID NO: 255 TAG AGC TAA TAC ATG SEQ ID NO:256 TGT ATT TAT TAG ATA SEQ ID NO: 257 TTA TTA GAT AAA AAA SEQ ID NO:258 TGG TTC ATT CAA ATT SEQ ID NO: 259 TTC AAA TTT CTG CCC SEQ ID NO:260 CTG CCC TAT CAA CTT SEQ ID NO: 261 AAC TTT CGA TGG TAG SEQ ID NO:262 TCG ATG GTA GGA TAG SEQ ID NO: 263 AGG GTT CGA TTC CGG SEQ ID NO:264 AGC CTG AGA AAC GGC SEQ ID NO: 265 AGA AAC GGC TAC CAC SEQ ID NO:266 CTA CCA CAT CCA AGG SEQ ID NO: 267 ATC CAA GGA AGG CAG SEQ ID NO:268 AAG GCA GCA GGC GCG SEQ ID NO: 269 AGG CGC GCA AAT TAC SEQ ID NO:270 CAA ATT ACC CAA TCC SEQ ID NO: 271 GAG GTA GTG ACA ATA SEQ ID NO:272 GTA ATT GGA ATG AGT SEQ ID NO: 273 TGG AAT GAG TAC AAT SEQ ID NO:274 CCT TAA CGA GGA ACA SEQ ID NO: 275 CAA GTC TGG TGC CAG SEQ ID NO:276 TAA TTC CAG CTC CAA SEQ ID NO: 277 AGC GTA TAT TAA AGT SEQ ID NO:278 TTA AAG TTG TTG CAG SEQ ID NO: 279 GTT GCA GTT AAA AAG SEQ ID NO:280 AGC TCG TAG TTG AAC SEQ ID NO: 281 AAA TTA GAG TGT TCA SEQ ID NO:282 GTG TTC AAA GCA GGC SEQ ID NO: 283 ATT AGC ATG GAA TAA T SEQ ID NO:284 GGT TCT ATT TTG TTG SEQ ID NO: 285 TGT TGG TTT CTA GGA SEQ ID NO:286 GTC AGA GGT GAA ATT SEQ ID NO: 287 TGA AAT TCT TGG ATT SEQ ID NO:288 TGA AGA CTA ACT ACT SEQ ID NO: 289 TAC TGC GAA AGC ATT SEQ ID NO:290 GTT TTC ATT AAT CA SEQ ID NO: 291 AAC GAA AGT TAG GG SEQ ID NO: 292GAT CAG ATA CCG TCG SEQ ID NO: 293 ACC GTC GTA GTC TTA SEQ ID NO: 294TAG TCT TAA CCA TAA SEQ ID NO: 295 ACC ATA AAC TAT GCC SEQ ID NO: 296TAT GCC GACT AGG GAT SEQ ID NO: 297 TCG GCA CCT TAC GAG SEQ ID NO: 298TAC GAG AAA TCA AAG SEQ ID NO: 299 AGT ATG GTC GCA AGG SEQ ID NO: 300GGC TGA AAC TTA AAG SEQ ID NO: 301 AGC CTG CGG CTT AAT SEQ ID NO: 302TTA ATT TGA CTC AAC SEQ ID NO: 303 AAA CTC ACC AGG TCC A SEQ ID NO: 304TGG AGT GAT TTG TCT SEQ ID NO: 305 TGT CTG CTT AAT TGC GAT SEQ ID NO:306 AAC AGG TCT GTG ATG SEQ ID NO: 307 TGT GAT GCC CTT AGA SEQ ID NO:308 GCG CGC TAC ACT GAC SEQ ID NO: 309 TTG CTC TTC AAC GAG

The present disclosure provides γPNA probes useful for the timelydetection and/or identification of sepsis-inducing pathogens without theneed of culturing the clinical specimens. These qualities are specificto the sequences of the optimized probes, however, one of skill in theart would recognize that other molecules with similar sequences couldalso be used. The γPNA probes provided herein comprise a sequence thatshares at least about 60-70% identity with a sequence described inTables 1-25. In another embodiment, the γPNA probe has a sequence thatshares at least about 71%, about 72%, about 73%, about 74%, about 75%,about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, or about 99% identity with thesequences of Tables 1-25 or complement thereof. The terms “identity” or“homology” or “similarity” refer to sequence relationships between twoγPNA sequences and can be determined by comparing a nucleotide positionin each sequence when aligned for purposes of comparison. The term“identity” refers to the degree to which nucleic acids are the samebetween two sequences. The term “homology” or “similarity” refers to therelatedness of two functionally-equivalent γPNA sequences.

The probe γPNA sequences also include functional fragments of thesequence provided in Tables 1-25 and sequences sharing certain sequenceidentities with those in Tables 1-25, as described above, provided theyfunction to specifically anneal to and identify sepsis-inducingpathogens. In one aspect, these fragment sequences have 1, 2, 3, 4, 5,or 6 less bases at either or both ends of the original sequences inTables 1-25. These shorter sequences are also within the scope of thepresent disclosure.

In addition, the γPNA sequences, including those provided in Tables 1-25and sequences sharing certain sequence identities with those in Tables1-25, as described above, can be incorporated into longer sequences,provided they function to specifically anneal to and identifysepsis-inducing pathogens. In one aspect, the longer sequences have 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 additional bases at either or both ends ofthe original sequences. These longer sequences are also within the scopeof the present disclosure.

The probe γPNA sequences are complementary to the target nucleic acidsequence. The probe γPNA sequences of the disclosure are optimal foridentifying numerous strains of a target nucleic acid, e.g.,Staphylococcus aureus.

Composition of γPNA Probes

In one embodiment, γPNA probes are used in the diagnosis of sepsis. γPNAprobes can be divided into two classes, γPNA capture probes and γPNAreporter probes.

PNA Capture Probes

In some embodiments, the γPNA capture probes are used for the capture,immobilization, and affinity purification of pathogenic genomic materialfrom the sample. In some embodiments, the γPNA capture probes aredesigned such that the probes' sequence binds only to one species ofpathogen. In some embodiments, the γPNA capture probes are designed suchthat the probes' sequence binds to more than one species of pathogen.

If targeted genomic material is present in the sample, the genomicmaterial will be captured and immobilized or bound by the γPNA captureprobe. If genomic material is not present; no genomic material will beimmobilized or bound by the γPNA capture probe. The capture and affinitypurification of the genomic content does not require the entire genomicfragment to be captured. Rather, a small portion of the target genomebeing captured constitutes the relevant portion of the genome beingcaptured. In one embodiment, the γPNA capture probes comprise of γPNAshaving one or more of the probe sequences listed in Tables 1-23.

The γPNA capture probes can be modified by one or more of thecharacteristics listed below. That is, the γPNA capture probes include,but are not limited to, the following embodiments.

In some embodiments, the γPNA capture probe sequences will bepre-immobilized onto a support substrate prior to introducing the sampleto be tested. In some embodiments, a “single γPNA capture probesequence” will be pre-immobilized onto a predefined location on asupport substrate. A “single γPNA capture probe sequence” is defined asonly one sequence for a single microbial pathogenic species, whichenables identification and quantification of the target pathogen. Forexample, a single species γPNA probe sequence encompassing only one ofthe probe sequences in Table 1 to enable capture of Staphylococcusaureus.

In another embodiment, “single γPNA capture probe sequence set,” allsequences to a single pathogen, can be pre-immobilized onto a predefinedlocation on a support substrate enabling identification andquantification of the target pathogen. “Single γPNA capture probesequence set” is defined as multiple sequences for a single microbialpathogenic species, which enables identification and quantification ofthe target pathogen. For example, a single species γPNA capture probesequence set encompassing two or more of the probe sequences in Table 1to enable capture of Staphylococcus aureus.

In another embodiment, one or more γPNA capture probe sequences for morethan one pathogen can be pre-immobilized onto a predefined location on asupport substrate with specificity, enabling identification andquantification of the pathogen subset of interest. For example, multipleγPNA capture probe sequences encompassing at least one probe sequencefrom both Table 1 and Table 2 to enable capture of Staphylococcus aureusand Enterococcus faecalis.

In some embodiments, the γPNA capture probes sequences in Tables 1-23include a moiety which enables them to be surface immobilized on asupport substrate. Immobilization of the γPNA capture probes sequenceson a support substrate can be achieved through various means such ascovalent binding protocols or non-covalent binding protocols. Commonbinding modalities/chemistries that can be used to immobilize the γPNAon the support substrate include, but are not limited to, COOH groups,NHS-ester groups, malemide chemistry, Click chemistry, streptavidin,thiol chemistry, and biotinylation. There are multiple additionalmethods which are commonly known to those skilled in the art.

In some embodiments the support substrate is coated with Avidin,Neutravidin, or Streptavidin to facilitate the immobilization of theγPNA capture probes sequences.

In some embodiments, the support substrate is one or more of the groupconsisting of a magnetic bead, a bead, a well, a plate, for example apolystyrene microtiter plate, a test tube, a stick, for exampledipstick, a plastic, a glass, and a chip or a biochip. In someembodiments, the support substrate is silicon based or coated witheither a semiconductive, conductive, or insulating material. In someembodiments, the support substrate includes metallic surfaces that arefunctionalized. In other embodiments the solid substrate may bemanufactured from polymers, nylon, nitrocellulose, polyacrylamide,oxides. In some embodiments, the solid support is manufactured frommultiple materials. In some embodiments, the surface of the supportsubstrate is coated with an aminosilane or any other commonly knownsurface treatments, such as epoxysilanes.

The γPNA capture probe lengths are considered to be substantiallyshorter than those typically used in similar applications due to theenhanced affinity of PNA probes in general and γPNA probes in particularwhen compared to DNA and RNA probes. The enhanced affinity is a resultof the neutral backbone and the pre-organization due to theγ-modification. Shorter probes in general are advantageous as they offersuperior sequence specificity.

In one embodiment, the γPNA capture probes have relatively shortnucleobase sequences, typically 5-30 bases in length. In anotherembodiment, the γPNA capture probes are 12-27 bases in length. Inanother embodiment, the γPNA capture probes are 15-24 bases in length.In another embodiment, the γPNA capture probes are 18-21 bases inlength.

In some embodiments, the γPNA capture probe may include moieties whichadd functionality to the probe itself. Examples include, but are notlimited to, binding molecules (such as biotin or haptens), spacergroups, linker groups, a hydrophobicity-changing group, acharge-inducing group, and a structural change-inducing group.

Examples of spacer groups include, but are not limited to,(ethylene)glycol, di(ethylene)glycol, tri(ethylene)glycol,poly(ethylene)glycol, 6-carbon linker, and 12 carbon linker.

Examples of linker groups include, but are not limited to, COOH group,NETS-ester group malemide chemistry, Click chemistry, streptavidin, andbiotinylation.

Examples of hydrophobicity-changing groups include, but are not limitedto, a naturally polar or charged side group or linker that decreaseshydrophobicity, such as as side groups mimicking those found onArginine, Histidine, Lysine, Aspartic Acid, Glutamic Acid, Serine,Threonine, Asparagine, and Glutamine, or a naturally apolar anduncharged side group or linker that increases hydrophobicity, such asside groups mimicking those found on Alanine, Valine, Isoleucine,Leucine, Methionine, Phenylalanine, Tyrosine, and Tryptophan.

Examples of charge-inducing groups include, but are not limited to, COOHgroup, NH₃ groups, OH groups, and metallic ions.

Structural change-inducing group induces a chemical modification in theγPNA capture probe's pseudo-peptide backbone to change the overallcharge of the PNA. Typical examples include a selection of positivelycharged or negatively charged amino acids to thereby alter the charge ofthe γPNA capture probe. In addition, small particles, small molecules,amino acids residues, small proteins or otherwise peptides may beincorporated, or conjugated along the backbone to alter the physicalcharacteristics of the γPNA capture probe, which would serve to eitheralter the affinity of the molecule or even its sequence specificity.Examples of structural change-inducing groups include, but are notlimited to, amino acid-based side chain, nanoparticle, small molecule orintercalating agent.

In some embodiment, the γPNA capture probe has an easily identifiablesignal induced at the capture site. Common signals include, but shallnot be limited to fluorescence, luminescence, FRET, colorimetric,calorimetric, interference patterns, pH, resistance/conductivity,enzymatic function and kinetics, protein structure, and electricalpotential. In some embodiments, detection of the presence of one or moretargeted genomic material is selected from electrical, massspectrometry, and/or precipitate.

γPNA Reporter Probes

The γPNA reporter probe is used to establish the presence, oralternatively the absence, of a targeted pathogen. γPNA reporter probesare designed to bind a conserved sequence region common among allbacteria. The γPNA reporter probe can be introduced regardless of thepresence or absence of the captured genomic material. In one embodiment,the γPNA reporter probes comprise of γPNAs having one or more of theprobe sequences listed in Tables 24-25. In one embodiment, the purposeof the γPNA reporter probe is to establish the presence of a targetpathogen through the presence of pathogenic genomic material at aparticular location. In some embodiments, the purpose of the γPNAreporter probe is to quantify the amount of target pathogen through itsgenomic material at a particular location.

In one embodiment, a single γPNA reporter probe sequence is used that iscommon, or universal, to the all of the potential targets. In anotherembodiment, multiple γPNA reporter probe sequences may be used wheretogether, as a group, they are universal to all or some of the potentialtarget pathogenic genomic material. In another embodiment, multiple γPNAreporter probe sequences may be used which may bind to multiplelocations along the target genomic material.

The γPNA reporter probe lengths are considered to be substantiallyshorter than those typically used in similar applications due to theenhanced affinity of PNA probes in general and γPNA probes in particularwhen compared to DNA and RNA probes. The enhanced affinity is a resultof the neutral backbone and the pre-organization due to theγ-modification. Shorter probes in general are advantageous as they offersuperior sequence specificity. In some embodiments, the γPNA reporterprobes have relatively short nucleobase sequences, typically 5-30 basesin length. In another embodiment the γPNA reporter probes have 12-27bases in length. In another embodiment, the γPNA capture probes are15-24 bases in length. In another embodiment, the γPNA capture probesare 18-21 bases in length.

The γPNA reporter probe contains a moiety that induces a signal. In oneembodiment, the γPNA reporter probe has a signal inducing capabilityselected from, but not limited to fluorophores, quantum dots, enzymes,conjugates, small molecules, chromophore, inorganic nanoparticles (suchas metals or semiconductors), conjugation enabling modifications,radioisotopes, and luminescent compounds. In some embodiments, the γPNAreporter probe is synthesized with a specific chemical moiety, whichlater enables the conjugation of a signal inducing compounds. Examplesof specific chemical moieties, but not limited to, are COOH groups,NETS-ester groups, malemide chemistry, Click chemistry, streptavidin,and biotinylation.

In some embodiments, the γPNA reporter probe may include moieties whichadd functionality to the probe itself. Examples include, but are notlimited to, binding molecules (such as biotin or haptens), spacergroups, linker groups, a hydrophobicity-changing group, acharge-inducing group, and a structural change-inducing group.

Examples of spacer groups include, but are not limited to,(ethylene)glycol, di(ethylene)glycol, tri(ethylene)glycol,poly(ethylene)glycol, 6-carbon linker, and 12 carbon linker.

Examples of linker groups include, but are not limited to, COOH group,NHS-ester group malemide chemistry, Click chemistry, streptavidin, andbiotinylation.

Examples of hydrophobicity-changing groups include, but are not limitedto, a naturally polar or charged side group or linker that decreaseshydrophobicity, such as Arginine, Histidine, Lysine, Aspartic Acid,Glutamic Acid, Serine, Threonine, Asparagine, and Glutamine, or anaturally apolar and uncharged side group or linker that increaseshydrophobicity, such as side groups mimicking those found on Alanine,Valine, Isoleucine, Leucine, Methionine, Phenylalanine, Tyrosine, andTryptophan.

Examples of charge-inducing groups include, but are not limited to, COOHgroup, NH₃ groups, OH groups, and metallic ions.

Structural change-inducing group induces a chemical modification in theγPNA reporter probe's pseudo-peptide backbone to change the overallcharge of the PNA. Typical examples include a selection of positivelycharged or negatively charged amino acids to thereby alter the charge ofthe γPNA reporter probe. In addition, small particles, small molecules,amino acids residues, small proteins or otherwise peptides may beincorporated, or conjugated along the backbone to alter the physicalcharacteristics of the γPNA reporter probe, which would serve to eitheralter the affinity of the molecule or even its sequence specificity.Examples of structural change-inducing groups include, but are notlimited to, amino acid-based side chain, nanoparticle, small molecule orintercalating agent.

However, in some embodiments, the γPNA reporter probe is not required.Rather, signal inducing agents such as DNA/RNA intercalating dyes, whichcan induce signals themselves, can be used. Several differentintercalating dyes are known, such as ethidum bromide and SYBR Green.These dyes are well established and usage of them is well known to thoseskilled in the art.

In some embodiments, where the γPNA reporter probe is not required, atarget pathogen may be PCR amplified with one or more of the primerscontaining fluorophores, quantum dots, enzymes, conjugates, smallmolecules, chromophore, inorganic nanoparticles (such as metals orsemiconductors), conjugation enabling modifications, radioisotopes, andluminescent compounds or with a specific chemical moiety, which laterenables the conjugation of a signal inducing compounds. Examples ofspecific chemical moieties, but not limited to, are COOH groups,NETS-ester groups, malemide chemistry, Click chemistry, streptavidin,and biotinylation. These methods are well established and known to thoseskilled in the art.

Carbon Linkers and Biotinylation

Carbon linkers serve different purposes depending on which type of γPNAprobe they are attached. γPNA capture probe utilize carbon linkers toremove issues due to potential steric hindrance between the surface ofthe support substrate and pathogenic genomic material.

In one embodiment, the γPNA capture probes' carbon linkers comprises ofat least one carbon. In another embodiment, the γPNA capture probes'carbon linkers comprises of 1-100 carbons. In another embodiment, theγPNA capture probes' carbon linkers comprises of 1-50 carbons. Inanother embodiment, the γPNA capture probes' carbon linkers comprises of1-25 carbons. In another embodiment, the γPNA capture probes' carbonlinkers comprises of 5-15 carbons.

γPNA reporter probe utilizes carbon linkers to eliminate issues ofsteric hindrance between the γPNA reporter probe and its signalingmoiety. In another embodiment, the γPNA reporter probes' carbon linkerscomprises of 1-100 carbons. In another embodiment, the γPNA reporterprobes' carbon linkers comprises of 1-50 carbons. In another embodiment,the γPNA reporter probes' carbon linkers comprises of 1-25 carbons. Inanother embodiment, the γPNA reporter probes' carbon linkers comprisesof 5-15 carbons.

The signal expressed by γPNA reporter probes can be amplified by havingmultiple biotinylation sites. In one embodiment, the γPNA reporterprobes' carbon-linker comprises of one or more biotinylation sites.

Methods for Diagnosing Sepsis Using γPNA Probes

In another embodiment, γPNA probes provide methods for diagnosingbacterial and fungal pathogens which induce sepsis.

In one embodiment, a γPNA capture probe, comprising one or more of theabove mentioned γPNA capture probe compositions, is combined andincubated with a sample from a person who is suspected of having sepsis.During the incubation, the γPNA capture probes will bind any genomicmaterial (dsDNA, ssDNA, or RNA) with the target sequence. In someembodiments, the mixture of γPNA capture probes and sample is heated tofacilitate invasion and binding of the γPNA capture probes to targetgenomic sequences.

In one embodiment, the method may consist of DNA amplification, forexample through PCR, of the genomic material in the sample.

In one embodiment, the genomic material in the sample is sheared.“Shearing” refers to shortening dsDNA, ssDNA, or RNA strands. Shearingcircumvents issues with DNA/RNA knotting/supercoiling due to the lengthof the bacterial genomic material. In some embodiments, the genomicmaterial is sheared to at least 10 kbp strands. In some embodiments, thegenomic material is sheared to about 10-500 bp. In other embodiments,the genomic material is sheared to 250-2,000 bp. In other embodiments,the genomic material is sheared from 1,000-10,000. In anotherembodiment, the genomic material is sheared to 5,000-50,000 bp. Shearingis well-known in the art and commercial kits are widely available.

In one embodiment, identification of the absence or presence of aparticular microbe through its genomic material requires a binary resultof capture or non-capture. In some embodiments, quantification of theload or copy number of pathogenic genomic material present in the samplecan be correlated to the number or amount of pathogenic genomic materialcaptured via the γPNA capture probe.

In one embodiment, a wash step is performed after incubation of the γPNAcapture probes and patient sample. Washing steps minimize unwanted,unbound, weakly bound, non-specifically bound target and othernon-target material from the vicinity of the γPNA capture probes.Washing steps are well established and known to those skilled in theart.

The capture of the genomic content does not enable identification byitself. Rather a detectable signal must be induced at the capture site.In one embodiment, the γPNA capture probe induces a signal upon bindingto the target sequence. The induced signal can be selected from, but notlimited to, fluorescence, luminescence, FRET, colorimetric,calorimetric, interference patterns, pH, resistance/conductivity,enzymatic function and kinetics, protein structure, and electricalpotential.

In alternate embodiment, after the affinity purification of the targetgenomic material by γPNA capture probe, a γPNA reporter probe,comprising one or more of the above mentioned γPNA reporter probecompositions, is introduced to the system. The γPNA reporter probe“invades” the immobilized target genomic material. In some embodimentsthe γPNA reporter probe contains a moiety which simplifies detection ofa signal. In some embodiments, the γPNA reporter probe is synthesizedwith such a signal inducing capability, which include, but not limitedto: fluorophores, quantum dots, enzymes, fluorescence, FRET, absorption,raman and/or SERS, chemiluminescence, bioluminescence, and scattering.

In some embodiments, the γPNA reporter probe is synthesized with aspecific chemical moiety, which later enables the conjugation of asignal inducing compounds. Examples of specific chemical moieties, butnot limited to, are: COOH groups, NETS-ester groups, malemide chemistry,Click chemistry, streptavidin, and biotinylation.

In some embodiments, the method of detecting γPNA reporter probe bindingto the target is selected from, but not limited to, electrical, massspectrometry, and/or precipitate.

In some embodiments, a wash step is performed to remove loosely boundγPNA reporter probes. These steps remove unwanted, unbound, weaklybound, non-specifically bound γPNA reporter probes from the system.

In some embodiments, the distance, in base pairs or bases, between theγPNA capture probe and γPNA reporter probe should be optimized to reducethe likelihood of DNA/RNA breakage between the two binding sites. Thedistance between the two probes should be sufficient such that theinvasion process is not hindered. In some embodiments, the probe sites,or target regions, are between about 10 to 100,000 bases apart. Inanother embodiment, the probe sites, or target regions, are betweenabout 50 to 75,000 bases apart. In another embodiment, the probe sites,or target regions, are between about 100 to 50,000 bases apart. Inanother embodiment, the probe sites, or target regions, are betweenabout 10,000 to 100,000 bases apart.

In some embodiments, the γPNA capture probes sequences do not identify aspecific species, but can identify a group of species. For example,Coagulase-Negative staphylococci (CoNS) encompasses a group ofStaphylococci species, which includes, but is not limited to,Staphylococcus capitis, Staphylococcus cohnii, Staphylococcusepidermidis, Staphylococcus haemolyticus, Staphylococcus hominis,Staphylococcus lugdunensis, Staphylococcus saprophyticus, Staphylococcuswarneri, Staphylococcus capitis. A pool of γPNA capture probe sequencescan be used to identify the CoNS group, see Table 18. For example, ifthe use of Pool 1 generates a positive signal, the signal indicates thatone or more of the above CoNS species is present. In some embodiments,the γPNA capture probes sequences are drawn from one or more pools fromTable 18.

Kit for Diagnosing Sepsis Using γPNA

In another embodiment, γPNA probes are used in kits for diagnosis ordetection of sepsis or determining the quantity of sepsis relatedgenomic material using γPNA. In one embodiment, the kit comprises aplurality of γPNA capture probes, wherein the γPNA capture probescomprise a sequence or the reverse-complementary sequence selected fromone or more of the sequences from Tables 1-23. The γPNA capture probeshaving any of the characteristics or conjugates previously described. Insome embodiments, the kit also comprises a plurality of γPNA reporterprobes, wherein the γPNA reporter probes comprise a sequence or thereverse-complementary sequence selected from one or more sequences fornTables 24-25, the γPNA reporter probes having any of the characteristicsor conjugates previously described.

EXEMPLIFICATION

The following examples describe embodiments, which are merelyillustrative and should not be construed as limiting in any way.

Example 1: Exemplary Overview

The following steps are a general overview of methods and material forone embodiment using γPNA probes to identify bacteria species in asample.

Step I: Loading Magnetic Beads with Capture Probes

With reference to FIG. 1A, a solution of excess capture probes 103 isincubated with Neutravidin coated 102 magnetic beads 101. Thebiotinylated γPNA capture probes naturally bind to the Neutravidincoated beads owing to the very high affinity of biotin to Neutravidin,which immobilizes the γPNA capture probes on the magnetic bead 104.After binding capture probes on the beads, excess probes are washed awayusing magnetic separation.

After rinsing, the beads are washed with solution of free biotin toblock the remaining unoccupied sites on the beads.

Step II: γPNA-Mediated Magnetic Extraction of Species-Specific DNA froma Mixture

With reference to FIG. 1B, a clinical sample of a patient with bothtarget DNA genomes 106 and non-targeted DNA genomes 107 is mixed withthe magnetic beads with bound γPNA capture probes 105 and heated at 50°C. The γPNA capture probes capture and immobilize the specific speciestarget onto the bead 108, if and only if the corresponding sequence ispresent in the sample. Extraction of the target DNA is then accomplishedby magnetically capturing the beads, and washing away the un-capturedbackground DNA.

Step III: Binding of γPNA Reporter Probes onto the Affinity PurifiedTargets

With reference to FIG. 1C, the magnetic beads with bound target DNA 109are re-suspended in a 50° C. solution with a large excess of the γPNAreporter probes 110. After γPNA reporter probes bind to the target 111,magnetic separation is used to wash away unbound γPNA reporter probes.If no target was present in the original sample, then no reporter probeswill remain in this step. Each probe is specific to a particularpathogen of interest, but is designed to bind a conserved sequenceregion, independent of the pathogen's subtypes.

Step IV: Binding of Reporter Enzyme onto Captured Targets

With reference to FIG. 1D, a commercial Neutravidin HRP conjugate(ThermoScientific) 113 is mixed with the magnetic beads, which havebound target DNA labeled with γPNA reporter probes 112. The HRPconjugate binds to the biotinylated γPNA reporter probe 114. Unboundenzymes are rinsed away using magnetic separation.

Step V: Reporter Substrate Addition and Optical Readout

With reference to FIG. 1E, a chemiluminescent HRP substrate 116 from acommercial ELISA kit (SuperSignal ELISA Femto, ThermoScientific) ismixed with the magnetic beads, which contain HRP conjugates 115. The HRPsubstrate is used to produce light signals indicating 117 the presenceof a target in a sample.

Additional optical methods of detection include, but are not limited to,fluorescence, FRET, quantum dots, absorption, raman and/or SERS,chemiluminescence, bioluminescence, and scattering. Other detectionmethods include, but are not limited to, mass spectrometry and/orprecipitate. Support substrates include but are not limited to any wellbased, dipstick, flow methods, chip, glass, bead, silicon, fibers,and/or paper.

Example 2: Identification of Staphylococcus aureus from a ClinicalSample

Staphylococcus aureus is detected in a clinical sample by adding thesample to a well or wells on a 96-well microplate, wherein each well iscoated with γPNA capture probes specific to Staphylococcus aureus. IfStaphylococcus aureus is present in the sample, a signal is detectedthat indicates possible sepsis infection in the patient. In analternative embodiment, positive and negative control samples are alsotested along with the clinical sample. The positive and negative controlsamples are also added to wells coated with γPNA capture probes specificto Staphylococcus aureus. The positive control is a sample known to haveStaphylococcus aureus. The negative control sample is known not to haveStaphylococcus aureus.

Materials and Methods

Streptavidin coated wells in 96-well microplates (Kaivogen Oy, Finland)are used as a solid support substrate. The microplate is pre-activatedwith γPNA capture probe(s), wherein the sequence of each capture probeis selected from one or more sequences from Table 1, which are sequencesspecific to Staphylococcus aureus. γPNA capture probes are synthesized(PNA Innovations, Inc., USA) to contain a biotin moiety on itsN-terminus, which enables binding of the γPNA capture probes to themicroplate well. Post-binding, the well is blocked with a biotin wash,which saturates all remaining biotin binding sites in the well.Post-blocking, the wells are thoroughly rinsed with a 0.2 micronfiltered 10 mM NaPi buffer (pH 7.0).

DNA from a clinical patient sample are isolated using a Wizard GenomicExtraction Kit (Promega, Inc., USA). After DNA isolation, the DNA issheared to a uniform length such as 10 kbp using a ‘G-Tube’ (Covaris,Inc. USA). Typical shearing protocol includes centrifuging extracted DNAsample for 60 sec at 8 krpm in an Eppendorf Minispin microcentrifuge(Eppendorf A G, Germany). Next, the DNA sample is concentrated and addedto the γPNA capture probe-activated well. To promote γPNA capture probeinvasion into the genomic target the well is heated to 60° C. for 30minutes in 10 mM NaPi (pH 7.0) with 15 mM NaCl, 0.05% Tween-20. Afterinvasion, the sample is washed to remove uncaptured DNA from the well.After the wash, γPNA reporter probes, which can also be biotinylated,are added to the well in 10 mM NaPi (pH 7.0) with 50 mM NaCl, 0.1% Tweento a final concentration of 1 uM. The γPNA reporter probes sequence isselected from one or more sequences from Table 24. The well is heatedusing the afore mentioned protocol and then washed to remove all unboundγPNA reporter probes.

Streptavidin conjugated HRP (VectorLabs, Inc., USA) is added to a finalconcentration of 1 ng/ml to each microplate well and incubated atroom-temperature for 30 min. Post-incubation, each well is washed toremove unbound Streptavidin conjugated HRP. Next, a substrate for HRP,such as SuperSignalFemto (Thermo-Scientific, USA), is added to each welland the emitted optical signal is read on a luminescence plate reader(GloMax 96, Promega, Inc., USA). The presence of Staphylococcus aureusin the clinical sample is indicated by an emitted optical signal.

Example 3: Determining if an Infection Arises from Coagulase NegativeStaphylococci (CoNS) or from Staphylococcus aureus

In one embodiment, γPNA probes are used to identify if a clinical sampleis infected with a CoNS species or Staphylococcus aureus. The clinicalsample originates from an individual who is deemed to have a possibleblood-borne infection. Staphylococcus aureus or a CoNS species aredetected in the sample by adding the sample to wells on a 96-wellmicroplate, wherein some wells are coated with γPNA capture probesspecific to Staphylococcus aureus and other wells are coated with γPNAcapture probes specific to CoNS species. A detectable signal inStaphylococcus aureus and/or CoNS species coated wells is indicative ofthe presence of that bacteria or bacterial family.

In an alternative embodiment, positive and negative control samples arealso tested along with the clinical sample. The positive and negativecontrol samples are also added to wells coated with γPNA capture probesspecific to Staphylococcus aureus or γPNA capture probes specific toCoNS species. The positive controls are samples known to haveStaphylococcus aureus and/or CoNS species. The negative control sampleis known not to have neither Staphylococcus aureus and CoNS species.

Methods and Materials

As previously described in Example 2, a 96-well microplate pre-coatedwith Streptavidin is used as a solid support substrate. Two differentγPNA capture probes or sets of capture probes (PNA Innovations, Inc.,USA) are added to one or more separate wells. The first well or set ofwells contain γPNA capture probes having one or more sequences selectedfrom Table 1, identified as CoNS−. The second well or set of wells,identified as CoNS+, has γPNA capture probes with sequences selectedfrom one of the pools listed in Table 18. The sequences within the poolare pre-mixed at equal-molar concentrations. The sequences found in eachpool in Table 18 are unique to a number of CoNS species, which include,but is not limited to, Staphylococcus capitis, Staphylococcus cohnii,Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcushominis, Staphylococcus lugdunensis, Staphylococcus saprophyticus,Staphylococcus warneri, Staphylococcus capitis. The γPNA capture probesare incubated on support substrate for 30 minutes to ensure properbinding. After binding of γPNA capture probes to the wells is completed,the wells are blocked with a biotin wash to saturate remaining biotinbinding sites. Post-blocking, the wells are rinsed with a 0.2 micronfiltered 10 mM NaPi buffer (pH 7.0).

DNA Amplification

DNA from a clinical patient sample is isolated using a Wizard GenomicExtraction Kit (Promega, Inc., USA). After DNA isolation, the DNA sampleis concentrated and amplified using Broad-Range PCR (specific to the 16sregion of bacteria) using Phusion DNA polymerase (New England Biolabs,Inc., USA).

The following primers are used for DNA amplification: Forward PrimerSequence: 5′ AGA GTT TGA TCC TGG CTC AG (SEQ ID NO: 310); Reverse PrimerSequence: 5′ ATC GGC TAC CTT GTT ACG ACT TC (SEQ ID NO: 311).

Both primers are mixed in equal-molar concentrations to a finalconcentration of 0.5 uM. dNTPs are added, likewise in equal-molarconcentrations, to a final concentration of 200 uM each. Phusion isadded to a final concentration of 2 u/100 ul with the recommend bufferand DNase/RNase-free water.

Thermocycling conditions are as follows:

-   -   1) 45 seconds at 98° C.×1 cycle    -   2) (15 seconds at 98° C., 30 seconds at 55° C., 75 seconds at        72° C.)×35 cycles    -   3) 5 minutes at 72° C.×1 cycle

Post-PCR processing, the amplified 16s region of the bacterial pathogenis isolated using the DNA Clean-uo kit (DNA Clean and Concentrator—5,Zymo Research, Inc., USA). After completing the DNA clean-up process,biotinylated γPNA reporter probes (PNA Innovations, Inc., USA) with oneor more sequence from Table 24 are added to the sample at a finalconcentration of 1.5 uM in 10 mM NaPi (pH 7.0) and heated for 30 min at60° C. The biotinylated γPNA reporter probes will invade the bacterial16s regions. Human DNA cannot be invade as it contains no 16s region.

γPNA Capture Probe Invasion Protocol

The sample is divided and added to both the CoNS− and the CoNS+ wellsand incubated with 10 mM NaPi (pH 7.0) with 5 mM NaCl, 0.05% Tween-20and heated for 30 min to 60° C. Upon completion of the incubationprocess, both wells are thoroughly rinsed to remove anyunbound/uncaptured DNA from each well.

Streptavidin Conjugated HRP Protocol

Streptavidin conjugated HRP (VectorLabs, Inc., USA) is added to a finalconcentration of 0.75 ng/ml to each well and incubated atroom-temperature for 30 min. The Streptavidin conjugated HRP binds tothe open biotin site displayed on the γPNA reporter probe. Postincubation, the wells are washed to remove unbound Streptavidinconjugated HRP from the well. Finally, a suitable substrate for HRP,such as SuperSignalFemto (Thermo-Scientific, USA) is added to each welland the emitted optical signal is read on a luminescence plate reader(GloMax 96, Promega, Inc., USA).

Results

If the clinical sample originally contained one or more CoNS species,the CoNS+ well produces a readily detectable optical signal. If theclinical sample originally contained Staphylococcus aureus, the CoNS−well produces a readily detectable optical signal. In the case whereneither one or more CoNS pathogens or Staphylococcus aureus is presentin the clinical sample, then both wells remain dark. Likewise, if one ormore CoNS species are present in addition to Staphylococcus aureus, boththe CoNS− and CoNS+ wells emit an optical signal, which is readilydetectable. In an alternative embodiment, when an optical signal isproduced from the sample, the signal is compared to the positive andnegative control samples to determine whether the signal indicates thepresence of Staphylococcus aureus and/or CoNS species.

Example 4: Discrimination of an Infection Arising from Two EnterococcalSpecies: Enterococcus faecalis and Enterococcus faecium

This example demonstrates the ability of γPNA probes to differentiatebetween two species of bacteria that belong to the same genus. A firstsample, which serves as a negative control, is produced by a healthysubject, i.e. does not contain pathogens. The second sample is from ansubject suspected of having a possible blood-borne infection. In analternative embodiment, positive control samples are also tested. Thepositive controls are a sample or samples known to have Enterococcusfaecalis and/or Enterococcus faecium.

Methods and Materials

Similar to the protocol of Example 2, a 96-well microplate is pre-coatedwith Streptavidin. In this example, two sets of two or more individualwells are coated with different γPNA capture probe, which have beensynthesized to contain a biotin moiety on its N-terminus. In the firstset of wells, γPNA capture probes with one or more sequences from Table2 are introduced into the wells, identified as E_faecalis. In the secondsets of wells, γPNA capture probes with one or more sequences from Table3 are introduced into the well, identified as E_faecium. Bindingprotocol, posting binding blocking, and wash steps from Example 3 willbe applied.

The two samples will be subject to the DNA amplification process andpost-PCR processing of Example 3.

Each sample is added to at least one well in each set. All wells will besubjected to the γPNA capture probes invasion protocol and excess DNAwash protocol described in Example 3.

After completing the excess DNA wash protocol, biotinylated γPNAreporter probes with one or more sequence from Table 24 are added toeach well at a final concentration of 1.5 uM in 10 mM NaPi (pH 7.0) andheated for 30 min at 60° C. After incubation the wells are washed toremove excess γPNA reporter probes.

Next the wells are subjected to the Streptavidin conjugated HRP protocoldetailed in Example 3.

The negative control wells for both E_faecalis and E_faecium should notyield an optical signal, beyond that which is expected for a samplewhich contains only non-target genomic material. If however, theclinical sample originally contained pathogens from the bacterialspecies Enterococcus faecalis, the E_faecium wells produce a clearlyidentifiable optical signal, whereas the E_faecium wells do not producea clearly identifiable optical signal. The reverse would be true if thepatient sample originally contained pathogens from the bacterial speciesEnterococcus faecium. Additionally, if the patient is infected by abacterial of a different species than Enterococcus faecalis orEnterococcus faecium, or by another pathogen, such as fungal or viral,both well types do not produce a positive readout signature.

Example 5: Discrimination of an Infection Arising from Candida Speciesor Aspergillus Species Fungal Pathogens Using Magnetic Beads

This example a clinical sample is tested for two different species ofbacteria using γPNA capture probes immobilized on a magnetic bead. Anegative control, as described in Example 4, is included to determinethe presence of the bacterial species in the clinical sample. Theclinical sample is from an individual who is deemed to have a possibleblood-borne infection that may be caused by Candida or Aspergillus. Inan alternative embodiment, positive control samples are also tested. Thepositive controls are a sample or samples known to have Candida and/orAspergillus.

Methods and Materials

In this embodiment, magnetic beads that have been pre-activated withamine active sites (DynaBeads, M-270 Amine, Inivtrogen, USA) areinitialized for the capture of either Candida or Aspergillus. To produceCandida specific magnetic beads, γPNA capture probes incorporatingequal-molar concentrations of probes specific to target one or moresequences in Tables 19-22 are covalently bound to the magnetic beadsutilizing the manufacturer's standard protocol. Likewise, magnetic beadsspecific to Aspergillus are produced by covalently binding the γPNAcapture probes specific to one or more target sequences in Table 23according to the manufacturer's standard protocol. In both cases, thiscaptures the γPNA capture probes to the surface through theirC-terminus. Each functionalized bead set is then placed into a separatemicrocentrigue tubes; one for the control sample, one for Candidaspecies, and one for Aspergillus species

The two samples are subjected to the DNA amplification process andpost-PCR processing discussed in Example 3. The following primers areused for DNA amplification:

Forward Primer Sequence: (SEQ ID NO: 312) 5′ AAA TCA GTT ATC GTT TAT TTGATA GT; Reverse Primer Sequence: (SEQ ID NO: 313) 5′ ATT CCT CGT TGA AGAGCA A.

The amplified genomic samples are added to their respectivemicrocentrifuge tubes and incubated for 10 min at 80° C. for 10 min in10 mM NaPi (pH 7.0) with 15 mM NaCl, 0.1% Tween-20. Upon completion ofthe incubation process, the magnetic beads are captured into a well orset of wells by a rare earth magnet. The well or sets of wells arerinsed to remove any unbound/uncaptured DNA from each magnetic bead.Post capturing targeted 18s regions, biotinylated γPNA reporter probeswith one or more sequence from Table 25 are added to the magnetic beadsat a final concentration of 2 μM in 10 mM NaPi (pH 7.0) with 5 mM NaCl,0.1% Tween-20 and heated at 75° C. for 15 min. After this incubationprocess, as before, the sample is rinsed/washed through magnetic beadimmobilization.

The magnetic beads are then subjected to the Streptavidin conjugated HRPprotocol detailed in Example 3.

Results

The optical density from negative control magnetic beads will benegligible and acts a baseline for comparison with the clinical sample.If the clinical sample contained pathogens from Candida, the magneticbeads that were functionalized with γPNA specific to Candida would yieldin increased absorbance compared to the control. If the clinical samplecontained pathogens from Aspergillus, the magnetic beads that werefunctionalized with γPNA specific to Aspergillus would yield inincreased absorbance. If the clinical sample was negative for bothCandida and Aspergillus, then both magnetic bead sets would have anoptical density measurement similar to the negative control.

Example 6: Identification of Escherichia coli from a Clinical Sample

A solid-support substrate contains the ability to specifically bind andcapture a pathogenic genomic target of interest. To achieve this, commonglass slides, which have been carboxylated (Xantec Bioanalytics,Germany), are utilized. The glass slides are pre-activated by spottingγPNA capture probes with one or more sequences from Table 4, which isspecific to E. coli. Binding of the γPNA capture probes to the glassslide is achieved through the N-terminus of the probe and accomplishedaccording to the manufacturer's protocol utilizing 750 nM γPNA captureprobe. Post binding, the glass slides are rinsed with a 0.2 micronfiltered 10 mM NaPi buffer (pH 7.0).

DNA from a clinical patient sample and a healthy patient (serving as anegative control) are isolated and amplified according to the methoddescribed in Example 3. The amplified DNA sample is added to the γPNAcapture probe spots on the glass slide. The DNA invasion process isperformed under the following conditions; 10 mM NaPi (pH 7.0) with 5 mMNaCl, 0.1% Tween-20, heated to 80° C. for 10 min. Post DNA invasion, thesample is washed. After the wash process, a DNA intercalating dye,Quant-iT PicoGreen (Life Technologies, USA) is added to the spottedsamples on the glass slide and incubated at room temperature in a darkroom for 20 min. Post incubation, the slide is washed thoroughly toremove non-intercalated dye.

After washing, the glass slide is imaged using an iXon EM-CCD camera(Andor Technology, UK) coupled with a 525 nm long-pass filter (EdmundOptics, USA), where the slide is excited via a 488 nm CW source(Coherent, USA). An optical signal attained from the spot where the γPNAcapture probe was initially immobilized would indicate the presence ofE. coli in the clinical sample.

Example 7: Identification and Quantification of Staphylococcusepidermidis from a Clinical Sample

In some embodiments, γPNA probes are used to identify a pathogen anddetermine the relative concentration of the pathogen in the clinicalsample.

Similar to that which was previously described, a 96-well microplatewhich pre-coated with Streptavidin. A single individual well or a set ofwells contain γPNA capture probes, which have been synthesized tocontain a biotin moiety on its N-terminus sequence. The γPNA captureprobes have one or more sequences selected from Table 5, whichspecifically target Staphylococcus epidermidis. The γPNA capture probebinding protocol of Example 3 is applied.

Known concentrations of Staphylococcus epidermidis (attained via ATCCA)is added to a pathogen-free sample. A calibration curve is created byusing eight different known concentration samples, ranging from 10° to10⁷ CFU/ml. After adding Staphylococcus epidermidis to the sample,genomic DNA is extracted using a Wizard Genomic Extraction Kit (Promega,Inc., USA).

The eight samples will be subject to the DNA amplification process andpost-PCR processing of Example 3.

After completing the DNA clean-up process, biotinylated γPNA reporterprobes with at least three different sequences from Table 24 are addedinto the sample at a final concentration of 2 uM (per each sequence) in10 mM Tris-HCl (pH 7.4) 0.05% and heated for 15 min to 75° C. MultipleγPNA reporter probes serve to amplify the number of active sitesintroduced into the 16s region. This incubation process is done for eachof the eight known concentration samples, individually, in γPNA captureprobe pre-activated wells. Upon completion of the incubation process,all wells are rinsed to remove any unbound/uncaptured DNA or PNA fromeach well.

Next, Streptavidin conjugated HRP (VectorLabs, Inc., USA) is added to afinal concentration of 1.5 ng/ml to each of the microplate wells andincubated at room-temperature for 15 min. The Streptavidin conjugatedHRP binds to the open biotin site displayed on the γPNA reporter probes.Post incubation, the wells are washed to remove unbound Streptavidinconjugated HRP from the well. Finally, a suitable substrate for HRP,such as SuperSignalFemto (Thermo-Scientific, USA) is added to each welland the emitted optical signal is read on a luminescence plate reader(GloMax 96, Promega, Inc., USA). The intensity signal is then plottedverse the known concentration which the clean sample was spiked with.This also serves to identify the saturation point of the system andlikewise the limit of detection of the system. Negative controls of justthe clean sample, which followed the same protocol as the eight knownsamples, are used to estimate the background signal.

DNA from the clinical patient sample is extracted and isolated using aWizard Genomic Extraction Kit (Promega, Inc., USA).

DNA is concentrated and the 16s bacterial region is amplified usingBroad-Range PCR (specific to the 16s region of bacteria) with thefollowing protocol with Phusion DNA polymerase (New England Biolabs,Inc., USA):

The sample is subjected to the DNA amplification process and post-PCRprocessing of Example 3.

After completing the DNA clean-up process, an optical signal from theclinical sample is generated using the same protocol used on the eightknown samples, discussed above.

The attained optical signal can then be compared to the previouslyproduced calibration curve, thereby enabling an estimation of thepathogen load of Staphylococcus epidermidis.

Example 8: Gel Shift Assay Demonstrating Binding of Staphylococcusaureus γPNA Probes to Target Genomic Material

The specificity of γPNA probes designed to target Staphylococcus aureuswas demonstrated by mixing Staphylococcus aureus targeted γPNA probeswith either a known sample having Staphylococcus aureus genomic materialor a known sample negative for Staphylococcus aureus genomic material(non-target genomic material). Binding of the γPNA probes toStaphylococcus aureus genomic material and lack of binding to non-targetgenomic material was measured by gel shift assays.

Methods and Materials

γPNA capture probes having sequences that target SEQ ID NO: 7, whichtargets Staphylococcus aureus, were incubated with either a sample ofStaphylococcus aureus genomic material or a sample having non-targetgenomic material.

γPNA reporters probes having sequences that target SEQ ID NO: 232, whichtargets a conserved sequence region common among all bacteria, wereincubated with either a sample of Staphylococcus aureus genomic materialor a sample having non-target genomic material.

A control sample of Staphylococcus aureus genomic material withoutincubation with γPNA probes was included in the assay. See FIG. 2.

The Staphylococcus aureus genomic material was ˜350 bp DNA fragmentsthat were amplified from Staphylococcus aureus (ATCC #43300).

After incubation, the samples were run on a 8% non-denaturing PAGE. Thegel was stained for DNA using SybrSafe genomic material intercalatingstain.

Results

As shown in FIG. 2, γPNA capture probes having sequences targeting SEQID NO: 7 were bound to Staphylococcus aureus genomic material afterincubation (Lane 2), as indicated by the band shift of Lane 2 whencompared to Lane 4, which was Staphylococcus aureus genomic materialwithout incubation with γPNA probes. Furthermore, Lane 3, which was γPNAcapture probes incubated with non-target genomic material, did not showa shift when compared to Lane 4. The shift in Lane 2 and lack of shiftin Lane 3, indicates that the γPNA capture probes were boundspecifically to the Staphylococcus aureus genomic material.

Similar results were seen with the γPNA reporters probes havingsequences targeting SEQ ID NO: 232. The γPNA reporters probes also boundspecifically to Staphylococcus aureus genomic material (FIG. 2, Lane 5).Lane 5, which contained γPNA reporters probes incubated withStaphylococcus aureus genomic material, shifts when compared to Lane 4.Additionally, γPNA reporters probes incubated with non-target genomicmaterial, Lane 6, does not shift, as compared to Lane 4. Thus, γPNAreporters probes bound specifically to Staphylococcus aureus genomicmaterial.

Example 9: Gel Shift Assay Demonstrating the Sequence Specificity ofStaphylococcus Aureus γPNA Capture Probes

The specificity of γPNA capture probes was demonstrated by comparing thebinding of γPNA capture probes targeting Staphylococcus aureus to asample known to have Staphylococcus aureus genomic material or to asample known to have Staphylococcus epidermidis genomic material.

Materials and Methods

γPNA capture probes with sequences targeted at SEQ ID NO: 7, whichtargets Staphylococcus aureus, was combined with a sample that was knowto have Staphylococcus aureus genomic material. The Staphylococcusaureus genomic material was obtained by PCR amplification of the 16sregion of Staphylococcus aureus DNA. Staphylococcus aureus genomicmaterial not incubated with γPNA capture probes was used as a control.

γPNA capture probes with sequences targeted at SEQ ID NO: 7 was alsocombined with a sample that was know to have Staphylococcus epidermidisgenomic material. The Staphylococcus epidermidis s genomic material wasobtained by PCR amplification of a portion of the 16s region ofStaphylococcus epidermidis DNA. This portion of the 16s region ofStaphylococcus epidermidis differs from the 16s region of Staphylococcusaureus by a 2 bp mismatch (indicated by underline in FIG. 3).Staphylococcus epidermidis genomic material not incubated with γPNAcapture probes was used as a control.

Results

Referring to FIG. 3, only Lane 2, which contains γPNA capture probesincubated with Staphylococcus aureus genomic material, showed a shiftwhen compared to Lane 1 and 3, both contain Staphylococcus aureusgenomic material not incubated with γPNA capture probes. The shiftindicates that the γPNA capture probes were bound to the Staphylococcusaureus genomic material. Conversely, the γPNA capture probes incubatedwith Staphylococcus epidermidis genomic material, Lane 4, did not show ashift when compared to Lanes 1 and 3. The lack of a shift in Lane 4indicates that the γPNA capture probes specifically targetsStaphylococcus aureus.

The specificity of the γPNA capture probes was demonstrated as a 2 bpmismatch prevented the Staphylococcus aureus target γPNA capture probesfrom binding to the Staphylococcus epidermidis 16s region.

OTHER EMBODIMENTS

Other embodiments will be evident to those of skill in the art. Itshould be understood that the foregoing detailed description is providedfor clarity only and is merely exemplary. The spirit and scope of thepresent invention are not limited to the above examples, but areencompassed by the following claims. The contents of all referencescited herein are incorporated by reference in their entireties.

What is claimed is:
 1. A method for detecting bacterial and/or fungalgenomic material in a sample comprising: (a) contacting a plurality ofγPNA capture probes to the sample, wherein the γPNA capture probescomprise at least one sequence selected from the group consisting of:SEQ ID NOS: 1-18, SEQ ID NOS: 19-22, SEQ ID NOS: 23-28, SEQ ID NOS:29-34, SEQ ID NOS: 35-38, SEQ ID NOS: 39-57, SEQ ID NOS: 58-72, SEQ IDNOS: 73-91, SEQ ID NOS: 92-94, SEQ ID NOS: 95-97, SEQ ID NOS: 98-110,SEQ ID NOS: 111-113, SEQ ID NOS: 114-117, SEQ ID NOS: 118-119, SEQ IDNOS: 120-121, SEQ ID NOS: 122-153, SEQ ID NOS: 154-166, SEQ ID NOS:167-190, SEQ ID NOS: 191-193, SEQ ID NOS: 194-196, SEQ ID NOS: 197-211,SEQ ID NOS: 212-215, SEQ ID NOS: 216-230, complementary sequencethereof, and functional equivalents thereof; (b) heating the γPNAcapture probes and the sample; (c) invading a plurality of bacterialand/or fungal genomic material by the γPNA capture probes; and (d)detecting a presence of one or more bacterial and/or fungal genomicmaterial.
 2. The method of claim 1, wherein the detecting step (d)comprises: (i) adding a plurality of γPNA reporter probes, wherein theγPNA reporter probes comprise at least sequence selected from the groupconsisting of SEQ ID NOS: 231-248 and SEQ ID NOS: 249-309, complementarysequence thereof, and functional equivalents thereof; (ii) heating theγPNA capture probes, the γPNA reporter probes, and the sample and (iii)invading of the γPNA reporter probes to the targeted genomic material,wherein the γPNA reporter probes are used to detect the targeted genomicmaterial.
 3. The method of claim 1, wherein the contacting step (a) ispreceded by an amplification step comprising an enzymatic amplificationof the genomic material in the sample.
 4. The method of claim 1, furthercomprising shearing the genomic material in the sample.
 5. The method ofclaim 1, wherein the γPNA capture probes are bound to a supportsubstrate.
 6. The method of claim 5, wherein a first carbon-linker bindsthe γPNA capture probes to the support substrate, and wherein the firstcarbon-linker comprises of at least three carbons.
 7. The method ofclaim 5, wherein the support substrate is selected from the groupconsisting of: a magnetic bead, a well, a plate, a test tube, a stick, aplastic slide, a glass slide, and a biochip.
 8. The method of claim 5,wherein the support substrate is coated with Avidin, Neutravidin, orStreptavidin.
 9. The method of claim 1, wherein the γPNA capture probescomprise one or more functional moiety selected from the groupconsisting of: a binding molecule, a spacer group, a linker group, ahydrophobicity-changing group, a charge-inducing group, and a structuralchange-inducing group.
 10. The method of claim 1, wherein the γPNAreporter probes comprise one or more functional moiety selected from thegroup consisting of: a binding molecule, a spacer group, a linker group,a hydrophobicity-changing group, a charge-inducing group, and astructural change-inducing group.
 11. The method of anyone of claim 1,wherein the γPNA probe comprises biotin or hapten.